It is likely that a cure for diabetes will involve multiple approaches, rather than one strategy being suitable for all patients. Diabetes is characterized by loss of beta-cells, but little is known about how to prevent that loss. Two logical approaches are to prevent beta-cell death, or to restore beta-cells after they are lost. This proposal will carry out fundamental studies that will inform both approaches. The long-term goal is to identify potential therapeutic targets for ameliorating beta-cell loss in diabetic patients. The approach is to use transgenic zebrafish as a model of neonatal diabetes and endoplasmic reticulum (ER) stress. The models include two different transgenic lines that express a human proinsulin containing a single gene mutation. Both mutations cause neonatal diabetes in humans. Such mutations prevent correct folding of the molecule and prevent it from being secreted. The misfolded proinsulin is retained in the ER, where it accumulates and eventually causes cell death. However, adult zebrafish, unlike mammals, have the ability to regenerate beta-cells. This provides a unique opportunity to identify genes that are required for maintaining beta-cell mass. Specific Aim 1 will study factors that positively and negatively influence beta-cell death. The focus is on characterizing the ER-stress response in beta-cells that accumulate misfolded proinsulin in the ER. The strategy is to quantify relative gene expression levels of conserved factors in the known vertebrate ER homeostasis pathways. Specific Aim 2 will compare differential gene expression in wild-type islets versus mutant, regenerative islets. Whole transcriptome analysis will identify genes that are required for maintenance and regeneration of beta-cell mass. This unbiased approach will also identify genes that are critical for maintaining ER homeostasis. The proposed research is innovative because it utilizes novel transgenic lines of zebrafish that will allow the in vivo study of beta-cell biology and regeneration, and because it uses disease-specific ablation. The proposed research is significant because it is expected to be a step forward in identifying therapeutic targets for maintaining or restoring beta-cell mass in human diabetic patients.